[go: up one dir, main page]

EP0626455B1 - Method of analysis, separation and micro-encapsulation of cell elements - Google Patents

Method of analysis, separation and micro-encapsulation of cell elements Download PDF

Info

Publication number
EP0626455B1
EP0626455B1 EP93830238A EP93830238A EP0626455B1 EP 0626455 B1 EP0626455 B1 EP 0626455B1 EP 93830238 A EP93830238 A EP 93830238A EP 93830238 A EP93830238 A EP 93830238A EP 0626455 B1 EP0626455 B1 EP 0626455B1
Authority
EP
European Patent Office
Prior art keywords
cell
separation
analysis
sub
microencapsulation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP93830238A
Other languages
German (de)
French (fr)
Other versions
EP0626455A1 (en
Inventor
Sergio Lucretti
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agenzia Nazionale per le Nuove Tecnologie lEnergia e lo Sviluppo Economico Sostenibile ENEA
Becton Dickinson Italia SpA
Original Assignee
Agenzia Nazionale per le Nuove Tecnologie lEnergia e lo Sviluppo Economico Sostenibile ENEA
Becton Dickinson Italia SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Agenzia Nazionale per le Nuove Tecnologie lEnergia e lo Sviluppo Economico Sostenibile ENEA, Becton Dickinson Italia SpA filed Critical Agenzia Nazionale per le Nuove Tecnologie lEnergia e lo Sviluppo Economico Sostenibile ENEA
Priority to DE69319715T priority Critical patent/DE69319715D1/en
Priority to EP93830238A priority patent/EP0626455B1/en
Publication of EP0626455A1 publication Critical patent/EP0626455A1/en
Application granted granted Critical
Publication of EP0626455B1 publication Critical patent/EP0626455B1/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • G01N15/1409—Handling samples, e.g. injecting samples
    • G01N2015/1411—Features of sheath fluids

Definitions

  • the invention relates to a method to select cell elements, including mono-, multi- or sub-cell elements, that is specially useful for vegetal cell treatments when flow-cytometry procedures are involved.
  • the detection methods of manipulated cells by fluorescent markers, vital dyes, molecular probes or antibodies, associated to the flow-cytofluorometry, allow virtually to use any type of cell or cell sub-structure through a qualitative and quantitative analysis of the fluorescent emission and the automatic quick separation of the interesting elements.
  • the cell elements having a fluid substrate as vehicle, are intercepted once at the time, and the signal quality and intensity analyzed.
  • the corresponding signal to any single cell element could be identified and separated from the other element signals by means of an electrical polarization and micro-drop fractionating mechanism of the cell flow, that is controlled by the vibrations of a piezoelectric crystal.
  • the electrical charged droplets, containing the actual interesting elements could be separated through deviating its from the normal trajectories when passing through a stationary electric field, and then collected under sterilization conditions into any suitable container.
  • vegetal biotechnology A number of examples of these techniques are found in animal and microbiology fields, while very limited uses concerned the vegetal biotechnology (1). This is mainly due to the brittleness features as well as to sizes of the vegetal cells, that poorly meet the requirements of the normal flowcytofluorometry instruments for animal cells, yet susceptible to move into a fluid (such as, for inst. within the blood). On the contrary, the vegetal cells are obtained from the plant tissues through the enzymic hydrolysis of the cellulose interconnecting structures, without which they lack rigidity and mechanical strength. Moreover, due to the presence of a large vacuole in the vegetal cell cytoplasm; which represents appr. the 90 percent of the total volume and implies an appr. 1000 time larger volume than the animal cell volume, a high cell breakage fraction occurs during the isolation procedure, and accordingly a limited recovery of the interesting cells usually results.
  • the inventor overcame the problems of the existing art by providing a method to analyze, sort and enencapsulate selected cell elements, such as single cells, sub-cell structures or multi-cell aggregates, from an inert substrate, which allows to isolate the cell elements as above at high efficiency level.
  • selected cell elements such as single cells, sub-cell structures or multi-cell aggregates
  • the use of such methods made very more effective the cell manipulation technology, associated with the flow-type cytofluoro- metry, specially when vegetal cells are involved.
  • the cell elements are resuspended in a fluid medium, comprising an inert material, having the ability to solidify under certain conditions.
  • this fluid medium showed an ability to be used in a flow-cytofluorometry procedure, through which the particles could be both qualitatively and quantitatively analyzed, then separated as microdroplets using a fractionation procedure; each droplet containing only one element, and, under proper conditions, solidifying instantaneously, though maintaining the integrity of each involved element.
  • the collected material is purified at a high level (90-99 percent of purity) and only contains the preselected elements through the cytoflurometry analysis, and therefore could be available for subsequent in vitro manipulations in order to obtain:
  • the ability of single cells to be manipulated within a solidified droplet considerably facilitates the recovery of excreta from the culture medium or the biochemically converted material from the same cells.
  • the process according to this invention allows to use more effectively the flow-type cytofluorometry as an instrument to analyze and separate vegetal cell as well as cell elements, by optimizing the recovery of unbroken structures into an inert matrix, which is perfectly compatible with, and advantageous for the cell growth.
  • the separated cells and the cell elements using the method of this invention maintain perfectly its integrity and are collected in a considerably higher percentage than the existing art methods, such as the unmodified analysis and separation procedure, and also they are able to be manipulated and/or to grow until the desired final product is obtained.
  • a scope of this invention is a method to analyze, separate and micro-encapsulate cell, sub cell or multicell elements, comprising to resuspend said elements in a fluid medium, at least comprising an inert substance, that is able to solidify in the presence of a catalyzer, being said catalyzer not comprised into said fluid medium; to feed said fluid medium to a container of the fluid to be supplied to a flow-type cytometer; this cytometer analysis comprising to fractionate said fluid medium in form of micro-sized droplets so that any droplet contains no more than only one of said elements; and to separate and collect said micro-sized droplets into containers wherein said solidification catalyzer is present.
  • said method to analyze, separate and micro-encapsulate cell elements comprises a treatment of said elements with fluorochromes before said resuspension procedure and said separation occurs following a fluore- scent emission from said elements.
  • said separation occurs by diffraction.
  • said fluid medium loading to the container of the flow-type cytometer fed fluid is carried out after having filtered the fluid medium.
  • said fluid medium comprises an isotonic salt solution and said inert solidifying material is selected from the product range as follows: alginate salts, agarose, agar-agar, carrageenan, gelatin, polyacrilamide, polystirene, dextran and precipitated metal hydroxides.
  • said solidifying material comprises sodium alginate and said catalyzer comprises a compound, which releases bi- or trivalent ions when in solution.
  • said sodium alginate solution is at a concentration in the range from 0.2 and 1.0 percent and said catalyzer comprises calcium chloride at a concentration range from 10 to 100 mM.
  • the sodium alginate con- centration is 0.5 percent and the calcium chloride concentration is 50 mM.
  • the cell elements have vegetal origin, and preferably are isolated from one of the following species: Nicotiana benthamiana, Solanum tuberosum and Citrus sinensis.
  • the cytofluorometer Fecstar Plus Becton Dickinson, San Jose, California USA
  • a fluid must circulate was properly and repeatedly washed out with a 70 percent EtOH solution; it is to be outlined that a high capacity, on-line absolute filter in the circuit to sterilize the sheat fluid was maintained in contact with EtOH for at least 1 hour.
  • Figure 1 shows a scheme of the process: the fluid medium 1, containing the inert solidifying substance for the micro-encapsulation, is delivered by means of the pressurized air 2 through the line 3 to the chamber of focusing 4.
  • the particles 5 to be analyzed and separated are delivered by means of the pressurized air 6 through the line 7 to the chamber of focusing 4.
  • the particles 5 are injected into the focusing chamber 4 by an injector 8 controlled by a pizoelectric crystal 9 for the controlled production of micro-drops.
  • a polarizing impulse 11 arrives to the focusing chamber 4; the particles go through a nozzle 12 and a laser signal 13.
  • the generated signal 14 goes back to the computer 10.
  • the polarized particles are deviated by plates 15 with a costant voltage of 2500 V and are seeded into the recipient 16 containing the solidification catalyst.
  • the used cell elements were isolated from plants belonging to the following species: Solanum tuberosum and Citrus sinensis ; however, other species could be successfully used.
  • Citrus sinensis protoplasts were isolated from "Tarocco" cultivar cell suspensions, which were obtained from nucellus callus, as described in (4).
  • Nicotiana benthamiana protoplasts were isolated from leaves of in vitro cultivated seedings, 20-30 day aged, according to the procedure described in (5).
  • the isolated protoplasts were dipped into 1 ⁇ g/ml of FDA sigma for minutes at 25°C. Following the acetate hydrolysis from cytoplasm esterases, free fluorescein was generated, which emitted a green-yellow fluorescence when excited by a blue ligth; this substance had a tendency to concentrate in the cell interior.
  • the cell elements were placed into an isotonic culture medium, lacking of calcium and magnesium ions, in order to safeguard its structure and/or vitality and loaded in the cytofluorometric analysis vessels according to known methods to anyone skilled in the art.
  • a 0.5 percent sodium alginate solution was prepared in a salt medium, free from calcium ions. This solution was loaded into the cytofluorometer feeding vessel and was the actual matrix, from which the droplets containing the cell elements were obtained.
  • the analysis nozzle was selected as adequate to the particles to be selected and separated (usually, the nozzle diameter must be three or four times larger than the particle diameter, by taking into account the need to maintain the fluid flow rate at a minimum, in order to guarantee a higher analysis stability, without negatively affecting a correct sample hydrodynamic focusing for a correct element-by-element analysis according to a single-cell based technique (fig.2).
  • sheat fluid flow was stabilized by removing any existing liquid and air bubble into the circuit by introducing EtOH at 70 percent into the focusing chamber.
  • the vibration piezoelectric source as well as the deflection plates, that allow to stabilize the temperature, were energized; the flow breaking point and the ⁇ average interval among the droplets were established; the correction factor to be applied between the time of the fluorescent emission of any object and the corresponding drop delay was calculated and input to the apparatus to synchronize the polarization impulse on the drop containing the element, separation of which was desired.
  • the correction factor ranged from 6.0 and 9.0.
  • the signal values of the interesting particles were defined and used as sorting gates.
  • the chromosome suspension were obtained from Vicia faba apical roots, after chemical synchronisation, formaldehyde fixation and mechanical grinding, as described in (2), but with less modifications.
  • the isolated chromosomes were dyed with 50 ⁇ g/ml propidium iodide (PI), that associated with the double strand nucleic acids and increased 10 times its orange- red fluorescence when excited by blue ligth.
  • PI propidium iodide
  • the PI-coloured chromosomes were excited with 200 mW at 514 nm.
  • the fluorescence signals were collected through photomultipliers equipped with specific pass band filters (535 nm for fluorescein and 630 nm for propidium).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Description

The invention relates to a method to select cell elements, including mono-, multi- or sub-cell elements, that is specially useful for vegetal cell treatments when flow-cytometry procedures are involved.
The modern biotechnologies made practical to manipulate the genetic properties of organisms, embryos and cells by stable introduction of exogeneous genes, that provide a new phenotype to recipient cells, such as resistance against patogenic agents or environmental stress, or capability to generate new metabolites. Also somatic hybridation or high-level of secondary metabolite induction techniques (2) are comprised in the cell manipulation methods.
When vegetal cells are involved, due to the totipotency thereof, regenerate plants could be obtained having new characteristics, which could be very largely and advantegeously used in the agronomy, industry and food production fields (3).
A main limiting reason in spreading the cell manipulation methods to a large extent is the problem to detect and select the manipulated cells, as often they result as a very low fraction of the whole cell population. In order to overcame such problem, cell lines are used as recipient cells, that are selected according to special requirements, such as the ability to be cultivated in the presence of selective toxic substances. However, the use of such "laboratory" cell lines is a further limit to apply these methods on agro-industrial scale.
The detection methods of manipulated cells by fluorescent markers, vital dyes, molecular probes or antibodies, associated to the flow-cytofluorometry, allow virtually to use any type of cell or cell sub-structure through a qualitative and quantitative analysis of the fluorescent emission and the automatic quick separation of the interesting elements. The cell elements, having a fluid substrate as vehicle, are intercepted once at the time, and the signal quality and intensity analyzed. The corresponding signal to any single cell element could be identified and separated from the other element signals by means of an electrical polarization and micro-drop fractionating mechanism of the cell flow, that is controlled by the vibrations of a piezoelectric crystal. The electrical charged droplets, containing the actual interesting elements, could be separated through deviating its from the normal trajectories when passing through a stationary electric field, and then collected under sterilization conditions into any suitable container.
A number of examples of these techniques are found in animal and microbiology fields, while very limited uses concerned the vegetal biotechnology (1). This is mainly due to the brittleness features as well as to sizes of the vegetal cells, that poorly meet the requirements of the normal flowcytofluorometry instruments for animal cells, yet susceptible to move into a fluid (such as, for inst. within the blood). On the contrary, the vegetal cells are obtained from the plant tissues through the enzymic hydrolysis of the cellulose interconnecting structures, without which they lack rigidity and mechanical strength. Moreover, due to the presence of a large vacuole in the vegetal cell cytoplasm; which represents appr. the 90 percent of the total volume and implies an appr. 1000 time larger volume than the animal cell volume, a high cell breakage fraction occurs during the isolation procedure, and accordingly a limited recovery of the interesting cells usually results.
The inventor overcame the problems of the existing art by providing a method to analyze, sort and enencapsulate selected cell elements, such as single cells, sub-cell structures or multi-cell aggregates, from an inert substrate, which allows to isolate the cell elements as above at high efficiency level. The use of such methods made very more effective the cell manipulation technology, associated with the flow-type cytofluoro- metry, specially when vegetal cells are involved.
The cell elements are resuspended in a fluid medium, comprising an inert material, having the ability to solidify under certain conditions.
Surprisingly this fluid medium showed an ability to be used in a flow-cytofluorometry procedure, through which the particles could be both qualitatively and quantitatively analyzed, then separated as microdroplets using a fractionation procedure; each droplet containing only one element, and, under proper conditions, solidifying instantaneously, though maintaining the integrity of each involved element.
The collected material is purified at a high level (90-99 percent of purity) and only contains the preselected elements through the cytoflurometry analysis, and therefore could be available for subsequent in vitro manipulations in order to obtain:
  • multicell structures (in the case of vegetal cells similar to the artificial seeds), surely generated from a single cell (cloning);
  • an isolation of subpopulation cells as well as single cells (clones) having a high metabolic activity and an ability to bio-chemically convert special substrates and/or to yield secondary metabolism products with high added value.
Moreover, the ability of single cells to be manipulated within a solidified droplet (that could be defined as chemical "microreactors") considerably facilitates the recovery of excreta from the culture medium or the biochemically converted material from the same cells.
The process according to this invention allows to use more effectively the flow-type cytofluorometry as an instrument to analyze and separate vegetal cell as well as cell elements, by optimizing the recovery of unbroken structures into an inert matrix, which is perfectly compatible with, and advantageous for the cell growth. This provides a clear improvement in developing methods of in vitro selection of vegetal elements from high agronomic value organisms by preventing the need of the availability of any laboratory strain or previously manipulated material, to which suitable characters were added in order to make the selection practicable (such, for inst., strength against antibiotics, or antimetabolites, etc.).
The separated cells and the cell elements using the method of this invention maintain perfectly its integrity and are collected in a considerably higher percentage than the existing art methods, such as the unmodified analysis and separation procedure, and also they are able to be manipulated and/or to grow until the desired final product is obtained.
Therefore this invention allows:
  • to optimize the isolation and the recovery of animal or vegetal intact cells (shock protection by the solidified material);
  • to obtain multi-cell clones and organized embrionic structures (artificial seeds, only as far as the vegetal cells are concerned);
  • to provide "cell microreactors" that are able to generate and/or to convert active secondary metabolites;
  • to isolate, grew and recover microorganism colonies from a single microorganism;
  • to encapsulate nuclei, Altmann's granules, chloroplasts and chromosomes for further cell manipulations and/or genetic conversions as well as for studies over its growth regulation and in vitro duplication mechanisms.
Therefore a scope of this invention is a method to analyze, separate and micro-encapsulate cell, sub cell or multicell elements, comprising to resuspend said elements in a fluid medium, at least comprising an inert substance, that is able to solidify in the presence of a catalyzer, being said catalyzer not comprised into said fluid medium; to feed said fluid medium to a container of the fluid to be supplied to a flow-type cytometer; this cytometer analysis comprising to fractionate said fluid medium in form of micro-sized droplets so that any droplet contains no more than only one of said elements; and to separate and collect said micro-sized droplets into containers wherein said solidification catalyzer is present.
According to a preferred embodiment of this invention, said method to analyze, separate and micro-encapsulate cell elements comprises a treatment of said elements with fluorochromes before said resuspension procedure and said separation occurs following a fluore- scent emission from said elements.
In an alternative embodiment according to this invention, said separation occurs by diffraction.
According to a preferred embodiment of this invention, said fluid medium loading to the container of the flow-type cytometer fed fluid is carried out after having filtered the fluid medium.
According to an embodiment of this invention said fluid medium comprises an isotonic salt solution and said inert solidifying material is selected from the product range as follows: alginate salts, agarose, agar-agar, carrageenan, gelatin, polyacrilamide, polystirene, dextran and precipitated metal hydroxides. Preferably, said solidifying material comprises sodium alginate and said catalyzer comprises a compound, which releases bi- or trivalent ions when in solution. More preferably said sodium alginate solution is at a concentration in the range from 0.2 and 1.0 percent and said catalyzer comprises calcium chloride at a concentration range from 10 to 100 mM. Most preferably, the sodium alginate con- centration is 0.5 percent and the calcium chloride concentration is 50 mM.
According to a preferred embodiment of this invention the cell elements have vegetal origin, and preferably are isolated from one of the following species: Nicotiana benthamiana, Solanum tuberosum and Citrus sinensis.
This invention will be described in the following by some explanatory, but not limiting examples with reference to the annexed figures, wherein:
  • fig. 1 is a schematic view of the flow-type cytofluoro metry system, the micro-droplet formation and the micro encapsulating procedure; and
  • fig.2 shows a diagram of micro-capsule dimension variation versus the analysis nozzle diameter.
    • EXAMPLE 1
    In order to allow a germ free collection of the interesting elements, the cytofluorometer (Facstar Plus Becton Dickinson, San José, California USA) section, wherein a fluid must circulate, was properly and repeatedly washed out with a 70 percent EtOH solution; it is to be outlined that a high capacity, on-line absolute filter in the circuit to sterilize the sheat fluid was maintained in contact with EtOH for at least 1 hour.
    Figure 1 shows a scheme of the process: the fluid medium 1, containing the inert solidifying substance for the micro-encapsulation, is delivered by means of the pressurized air 2 through the line 3 to the chamber of focusing 4. At the same time, the particles 5 to be analyzed and separated are delivered by means of the pressurized air 6 through the line 7 to the chamber of focusing 4. The particles 5 are injected into the focusing chamber 4 by an injector 8 controlled by a pizoelectric crystal 9 for the controlled production of micro-drops. From a computer 10 a polarizing impulse 11arrives to the focusing chamber 4; the particles go through a nozzle 12 and a laser signal 13. The generated signal 14 goes back to the computer 10. The polarized particles are deviated by plates 15 with a costant voltage of 2500 V and are seeded into the recipient 16 containing the solidification catalyst.
    The used cell elements were isolated from plants belonging to the following species: Solanum tuberosum and Citrus sinensis; however, other species could be successfully used.
    Particularly, the Citrus sinensis protoplasts were isolated from "Tarocco" cultivar cell suspensions, which were obtained from nucellus callus, as described in (4).
    The Nicotiana benthamiana protoplasts were isolated from leaves of in vitro cultivated seedings, 20-30 day aged, according to the procedure described in (5).
    The isolated protoplasts were dipped into 1 µg/ml of FDA sigma for minutes at 25°C. Following the acetate hydrolysis from cytoplasm esterases, free fluorescein was generated, which emitted a green-yellow fluorescence when excited by a blue ligth; this substance had a tendency to concentrate in the cell interior.
    The cell elements were placed into an isotonic culture medium, lacking of calcium and magnesium ions, in order to safeguard its structure and/or vitality and loaded in the cytofluorometric analysis vessels according to known methods to anyone skilled in the art.
    In order to allow the encapsulating process, a 0.5 percent sodium alginate solution was prepared in a salt medium, free from calcium ions. This solution was loaded into the cytofluorometer feeding vessel and was the actual matrix, from which the droplets containing the cell elements were obtained.
    The essential parameters to separate cell and sub-cell elements are summarized in the table 1 (see p.12).
    The analysis nozzle was selected as adequate to the particles to be selected and separated (usually, the nozzle diameter must be three or four times larger than the particle diameter, by taking into account the need to maintain the fluid flow rate at a minimum, in order to guarantee a higher analysis stability, without negatively affecting a correct sample hydrodynamic focusing for a correct element-by-element analysis according to a single-cell based technique (fig.2).
    Essential Parameters To Separate Cell And Sub-Cell Elements
    Particles (larger size, µm) Nozzle diameter analysis (µm) Sheat fluid composition Sheat fluid pressure (psi) Vibration frequency (kHz) Amplitude frequency (V)
    chromosomes (2-10) 65 (60-100) KCl 80mM NaCl 20mM Na alginate 0.5% (w/v)
    pH 7.2    		 19.5 (21-15) 28 (30-12) 0.5 (0.5-2.0)
    protoplasts (30-200) 200 (100-500) KCl
    NaCl H2PO4
    Mannitol 0.3M Na alginate 0.5% (w/v)    		 4 (6-2.5) 6.1 (2-12) 2 (0.5-25)
    The test variability ranges are reported in brackets.
    Furthermore the sheat fluid flow was stabilized by removing any existing liquid and air bubble into the circuit by introducing EtOH at 70 percent into the focusing chamber.
    Then the vibration piezoelectric source as well as the deflection plates, that allow to stabilize the temperature, were energized; the flow breaking point and the λ average interval among the droplets were established; the correction factor to be applied between the time of the fluorescent emission of any object and the corresponding drop delay was calculated and input to the apparatus to synchronize the polarization impulse on the drop containing the element, separation of which was desired. Normally the correction factor ranged from 6.0 and 9.0. The signal values of the interesting particles were defined and used as sorting gates.
    A drop collecting vessel, containing a culture medium and 50 mM of CaCl2 as solidification catalyzer was fitted.
    The separation procedure was then started, by checking periodically that the flow rate was stable and the breaking point unchanged in order to detect immedia- tely any air bubble or on-line absolute filter obstruction occurrence. The summarizing data are shown in the table 2.
    Summary of the cell parameters in the micro-encapsulation procedure
    Particle starting concentr. (part/ml) Detecting parameter Sorting efficiency (%) Plating efficiency (%) Separated Cell Nr. p.Vessel Collect. Substr.
    Chromosomes 106 Dna (propidium iodide, 50 ug/ml) 99 = 104-105 LB + CaCl 2 50 mM
    Protoplasts 104-105 Viabiity (FDA 1 ug/ml) 95 (90-95) Encapsul. 1-105 Culture medium CaCl 2 50 mM
    A=90-60
    N=60-40
    Not encapsul.
    A=50-30
    N=30-0
    No toxic or disruptive effects were detected in the micro-encapsulating process due to the analyzed cell elements.
    EXAMPLE 2: Chromosome separation
    The chromosome suspension were obtained from Vicia fabaapical roots, after chemical synchronisation, formaldehyde fixation and mechanical grinding, as described in (2), but with less modifications.
    The isolated chromosomes were dyed with 50 µg/ml propidium iodide (PI), that associated with the double strand nucleic acids and increased 10 times its orange- red fluorescence when excited by blue ligth. The PI-coloured chromosomes were excited with 200 mW at 514 nm. The fluorescence signals were collected through photomultipliers equipped with specific pass band filters (535 nm for fluorescein and 630 nm for propidium).
    The concerned parameters are summarized in the previous tables 1 and 2.
    REFERENCES
  • (1) Weaver J.C., Bliss J.G., Powell K.T, Harrison G.I. and Williams G.B., "Rapid clonal growth measurements at the single-cell level: gel, microdroplets and flow cytometry", BIO/TECHNOLOGY, 9, 873-77, 1991.
  • (2) Lindsey K. and Yeoman M.M., "Novel experimental systems for studying the production of secondary metabolites by plant tissue culture" from " Plant Biotechnology", edited by Mantell S.H. and Smith H., Cambridge Univ. press, 39-66, 1986.
  • (3) Lister A., "G4Flowcitometry for selection of plant cells in vitro" from "Plant cell line selection procedures and applications", edited by Dix P.J., VCH press, 39-50, 1990.
  • (4) S. Kobayashi, H. Ucimaja and 1. Ikeda, "Plant regeneration from "Trovita" orange protoplasts", Jpn. J.Breeding, 33, 119, 1983.
  • (5) Alfonso C.L., Harkins K.R., Thomas Compton M.A., Krejci A. E. and galbraith D.W., " Selection of somatic hybrid plants in Nicotiana through fluorescence activated sorting of protoplasts", Bio/Technology, 3, 811-816, 1985.

    • Claims (10)

    1. A method of analysis, separation and micro encapsulation of cell, sub-cell or multi-cell elements characterized in comprising the steps as follows:
      to resuspend said elements in a fluid medium, comprising at least an inert material that is able to solidify in presence of a catalyzer; being said catalyzer not comprised in said fluid medium;
      to load said fluid medium into a fluid feeding vessel of a flow-type cytometry apparatus;
      to carry out a cytometry analysis, comprising to fractionate said fluid in microdroplets, each one of which contains no more than only one element;
      to separate and collect said microdroplets within vessels containing said solidification catalyzer.
    2. A method of analysis, separation and microencapsulation of cell, sub-cell or multi-cell elements according to the claim 1, wherein said resuspension follows a said element treatment with fluorochromes and said separation results from a fluorescence emission of said elements.
    3. A method of analysis, separation and microencapsulation of cell, sub-cell or multi-cell elements according to the claim 1, wherein said separation results from a diffraction.
    4. A method of analysis, separation and microencapsulation of cell, sub-cell or multi-cell elements according to any previous claim, wherein said loading step follows a filtering operation.
    5. A method of analysis, separation and microencapsulation of cell, sub-cell or multi-cell elements according to any previous claim, wherein said fluid medium comprises an isotonic salt solution, and said inert solidifying material is selected from the following range: alginate salts, agarose, agar-agar, carrageenan, gelatin, polyacrilamide, polystirene, dextran and precipitated metal hydroxides.
    6. A method of analysis, separation and microencapsulation of cell, sub-cell and multi-cell elements according to the claim 5, wherein said inert solidifying material comprises sodium alginate, and said catalyzer comprises a compound releasing bivalent or trivalent ions when solubilized.
    7. A method of analysis, separation and microencapsulation of cell, sub-cell or multi-cell elements according to the claim 6, wherein said sodium alginate solution is at concentration ranging from 0.2 to 1.0 percent, and said catalyzing compound comprises calcium chloride at a concentration ranging from 10 to 100 mM.
    8. A method of analysis, separation and microencapsulation of cell, sub-cell or multi-cell elements according to the claim 7, wherein the said sodium alginate concentration is 0.5 percent and said calcium chloride concentration is 50 mM.
    9. A method of analysis, separation and microencapsulation of cell, sub-cell or multi-cell elements according to any previous claim, wherein said elements are from a vegetal source.
    10. A method of analysis, separation and microencapsulation of cell, sub-cell or multi-cell elements according to the claim 9, wherein said vegetal elements are isolated cells from one of the following species: Nicotiana benthamiana, Solanum tuberosum, Citrus sinensis, or sub-cell elements thereof, including chromosome.
    EP93830238A 1993-05-28 1993-05-28 Method of analysis, separation and micro-encapsulation of cell elements Expired - Lifetime EP0626455B1 (en)

    Priority Applications (2)

    Application Number Priority Date Filing Date Title
    DE69319715T DE69319715D1 (en) 1993-05-28 1993-05-28 Methods for the analysis, separation and microencapsulation of cellular elements
    EP93830238A EP0626455B1 (en) 1993-05-28 1993-05-28 Method of analysis, separation and micro-encapsulation of cell elements

    Applications Claiming Priority (1)

    Application Number Priority Date Filing Date Title
    EP93830238A EP0626455B1 (en) 1993-05-28 1993-05-28 Method of analysis, separation and micro-encapsulation of cell elements

    Publications (2)

    Publication Number Publication Date
    EP0626455A1 EP0626455A1 (en) 1994-11-30
    EP0626455B1 true EP0626455B1 (en) 1998-07-15

    Family

    ID=8215169

    Family Applications (1)

    Application Number Title Priority Date Filing Date
    EP93830238A Expired - Lifetime EP0626455B1 (en) 1993-05-28 1993-05-28 Method of analysis, separation and micro-encapsulation of cell elements

    Country Status (2)

    Country Link
    EP (1) EP0626455B1 (en)
    DE (1) DE69319715D1 (en)

    Families Citing this family (5)

    * Cited by examiner, † Cited by third party
    Publication number Priority date Publication date Assignee Title
    GB2316081B (en) * 1996-08-12 2001-05-09 Bio Dot Ltd Dispensing of particles
    WO2000008212A1 (en) 1998-08-07 2000-02-17 Cellay, Llc Gel microdrops in genetic analysis
    US6400453B1 (en) * 1998-08-21 2002-06-04 Union Biometrica, Inc. Instrument for selecting and depositing multicellular organisms and other large objects
    AU2606301A (en) * 1999-12-29 2001-07-09 Union Biometrica, Inc. High viscosity sheath reagent for flow cytometry
    US6482652B2 (en) * 2000-03-23 2002-11-19 The Board Of Trustees Of The Leland Stanford Junior University Biological particle sorter

    Family Cites Families (1)

    * Cited by examiner, † Cited by third party
    Publication number Priority date Publication date Assignee Title
    EP0022434B1 (en) * 1979-06-27 1985-08-28 Peter Brodelius Catalysts for the production and transformation of natural products having their origin in higher plants, process for production of the catalysts, and use thereof

    Also Published As

    Publication number Publication date
    EP0626455A1 (en) 1994-11-30
    DE69319715D1 (en) 1998-08-20

    Similar Documents

    Publication Publication Date Title
    Gupta et al. Somatic proembryo formation and transient expression of a luciferase gene in Douglas fir and loblolly pine protoplasts
    TWI457439B (en) In vitro methods for the induction and maintenance of plant cell lines as single suspension cells with intact cell walls, and transformation thereof
    deJong et al. Mammalian artificial chromosome pilot production facility: large‐scale isolation of functional satellite DNA‐based artificial chromosomes
    US11718845B2 (en) Methods for increasing gene-editing frequencies in maize cells
    CN113444678A (en) Method for preparing single cell nuclear suspension, single cell sequencing method and kit
    WO2018143480A1 (en) Method for introducing substance into plant
    EP0626455B1 (en) Method of analysis, separation and micro-encapsulation of cell elements
    Hammatt et al. Selection of plant heterokaryons from diverse origins by flow cytometry
    Meehan et al. Lhcb transcription is coordinated with cell size and chlorophyll accumulation (studies on fluorescence-activated, cell-sorter-purified single cells from wild-type and immutans Arabidopsis thaliana)
    US4300310A (en) Identification and sorting of plant heterokaryons
    Galbraith Cytometry and plant sciences: a personal retrospective
    Louzada et al. Preparation and fusion of Citrus sp. microprotoplasts
    Ramulu et al. Cremart: a new chemical for efficient induction of micronuclei in cells and protoplasts for partial genome transfer
    US10480001B2 (en) Method for the generation of a monoclonal plant cell line
    JP2602010B2 (en) Method for the maintenance and propagation of a deleted non-infectious plant virus genome or DNA and regenerated plant cell, plant callus or plant tissue
    SUGINIURA et al. Gene transfer by electroporation into protoplasts isolated from mulberry calli
    Saunders et al. Chemically induced fusion of plant protoplasts
    KR20220007616A (en) Methods, systems and apparatus for screening and propagation of plant material
    Galbraith Flow cytometric analysis and sorting of somatic hybrid and transformed protoplasts
    Pattanavibool et al. Interspecies protoplast fusion in Larix: comparison of electric and chemical methods
    Burlion et al. Electrical fusion of protoplasts from sycamore mutants and hybrid selection on hypoxanthine-aminopterin-thymidine (HAT) medium
    杉村順夫 et al. Gene transfer by electroporation into protoplasts isolated from mulberry calli.
    Cocking Plant protoplasts as genetic systems
    Dodds Fusion of plant protoplasts
    Lyznik et al. Polyethylene glycol-mediated DNA uptake into maize protoplasts

    Legal Events

    Date Code Title Description
    PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

    Free format text: ORIGINAL CODE: 0009012

    AK Designated contracting states

    Kind code of ref document: A1

    Designated state(s): CH DE FR GB LI NL

    17P Request for examination filed

    Effective date: 19941209

    17Q First examination report despatched

    Effective date: 19970226

    GRAG Despatch of communication of intention to grant

    Free format text: ORIGINAL CODE: EPIDOS AGRA

    GRAG Despatch of communication of intention to grant

    Free format text: ORIGINAL CODE: EPIDOS AGRA

    GRAH Despatch of communication of intention to grant a patent

    Free format text: ORIGINAL CODE: EPIDOS IGRA

    GRAH Despatch of communication of intention to grant a patent

    Free format text: ORIGINAL CODE: EPIDOS IGRA

    GRAA (expected) grant

    Free format text: ORIGINAL CODE: 0009210

    AK Designated contracting states

    Kind code of ref document: B1

    Designated state(s): CH DE FR GB LI NL

    PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

    Ref country code: NL

    Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

    Effective date: 19980715

    Ref country code: LI

    Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

    Effective date: 19980715

    Ref country code: FR

    Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

    Effective date: 19980715

    Ref country code: CH

    Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

    Effective date: 19980715

    REG Reference to a national code

    Ref country code: CH

    Ref legal event code: EP

    REF Corresponds to:

    Ref document number: 69319715

    Country of ref document: DE

    Date of ref document: 19980820

    PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

    Ref country code: DE

    Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

    Effective date: 19981016

    NLV1 Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act
    EN Fr: translation not filed
    REG Reference to a national code

    Ref country code: CH

    Ref legal event code: PL

    PLBE No opposition filed within time limit

    Free format text: ORIGINAL CODE: 0009261

    STAA Information on the status of an ep patent application or granted ep patent

    Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

    PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

    Ref country code: GB

    Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

    Effective date: 19990528

    26N No opposition filed
    GBPC Gb: european patent ceased through non-payment of renewal fee

    Effective date: 19990528